Natural killer (NK) cells play a pivotal role in host immunity against different malignancies, including pancreatic ductal adenocarcinoma (PDAC). Our study aimed to evaluate the antitumor effects of NK cell-based adoptive transfer immunotherapy for PDAC in an orthotopic mouse model. Orthotopic Kras LSL-G12D p53 LSL-R172H Pdx1-Cre (KPC) mice were used to evaluate the therapeutic efficacy. Mouse NK cells (LNK cells)(1× 10 6) were intravenously injected to tumor-bearing mice once a week for 3 weeks. MRI measurements (tumor volume and apparent diffusion coefficient (ADC) values) and survival were compared between control and LNK treated tumors. Flow cytometry and enzyme-linked immunosorbent assay (ELISA) were used to determine LNK cells cytotoxicity and IFN-γ level, respectively. LNK cells can produce a higher level of IFN-γ and more effectively lyse PDAC cells compared with spleen NK cells in vitro. LNK-cell adoptive transfer therapy elicited potent in vivo antitumor activity, resulting in delayed tumor growth (P= 0.033) in KPC mice. The ADC values at the last timepoint ((0.94±0.06)× 10-3 mm 2/s) were significantly higher than that at first timepoint ((0.75±0.04)× 10-3 mm 2/s) in treated tumors (P< 0.001). ADC values were significantly different between control group and treated tumors at the last time point ((0.75±0.09)× 10-3 mm 2/s vs (0.94±0.06)× 10-3 mm 2/s, P= 0.004) in KPC mice. Our data demonstrate the potential of NK cell-based adoptive transfer immunotherapy for PDAC treatment.